Synthesis of structural analogues of Reversan by ester aminolysis: an access to pyrazolo[1,5-a]pyrimidines from chalcones

Reversan, a multidrug resistance-associated protein (MRP1) inhibitor described more than a decade ago, is a commercial drug (CAS: 313397-13-6) that has a high price and is six to eight times more potent than known drug transporter inhibitors. However, to date, a complete route for synthesizing pyrazolo[1,5-a]pyrimidine-based Reversan is yet to be published. Herein, the silica gel-mediated synthesis of Reversan and a novel family of its structural analogues (amides) via the microwave-assisted amidation reaction of 3-carboethoxy-5,7-diphenylpyrazolo[1,5-a]pyrimidine (ester) with primary amines is reported. Moreover, a set of this ester-type precursor was obtained using the NaF/alumina-mediated reaction of 5-amino-3-carboethoxy-1H-pyrazole with chalcones, implying a final removal of H2 using Na2S2O8. Both esters and amides were obtained in high yields using heterogeneous catalyst and solvent-free, highly efficient, and scalable synthetic protocols.


Introduction
Intrinsic or acquired multidrug resistance is one of the leading causes of treatment failure in human malignancies; thus, nding new ways to solve this problem has attracted special attention from chemists, biologists, pharmacists, and related professionals. Molecular-level investigations of cancer multidrug resistance have revealed that two ATP-binding cassette transporters cause resistance in tumor cells: P-glycoprotein and the multidrug resistance-associated protein (MRP1). 1,2 The overexpression of MRP1 in almost all tumor types (e.g., lung, melanoma, sarcoma, neuroblastoma, head, and breast) lowers the intracellular drug concentration. [3][4][5] About this, Burkhart et al. 6 reported a way to overcome MRP1 activity using Reversan, a pirazolo[1,5-a]pyrimidine (PP) derivative having an N-(3-morpholinopropyl)carboxamide group at position 3 and two phenyl rings at positions 5 and 7, which is commercially available but has a high price (Fig. 1a).
On the other hand, ester aminolysis is possibly the least common method for forming amides, one of the most appreciated functional groups in biologically relevant compounds. [24][25][26][27][28] The low recurrence of direct ester amidation is possibly because the reactions require high temperatures. Under this condition, substrates are susceptible to decomposition or easy evaporation, especially liquid ones, leading to a loss of mass efficiency or low reaction yields. [24][25][26] The usual methods for preparing amides involve reacting carboxylic acids or acylating agents (i.e., acid anhydrides, acid chlorides, and acids with additives) with amines. However, most of these methods require excess amine, catalysts, coupling reagents, solvents excess, or high temperatures with prolonged reaction times, leading to protocols with a poor atom economy, generating toxic wastes, and spending energy. [24][25][26][27][28][29] Thus, the solventfree direct amidation reaction of carboxylic acids or esters by heterogeneous catalysis using silica or alumina is a suitable method, though little-used, to efficiently access amides due to the value and easy disposal of these solids (Scheme 2a). [29][30][31][32][33] Due to the biological importance of pyrazolo[1,5-a] pyrimidines [10][11][12][13] and the amide functional group, [24][25][26][27][28] there are several examples of synthesis of this N-heterocycle substituted at position 3 with the carboxamide group. However, when the respective ester is used as a starting reagent, the synthesis proceeds by hydrolysis and subsequent amidation under conditions that imply coupling agents, further reaction steps, and poor yields (Scheme 2b). [34][35][36] Pondering these ndings and the relevance of reversan, we envisioned that this amide and a novel family of its structural analogues (PPs 5a-h) could be synthesized through the direct amidation reaction of ethyl-5,7diphenylpyrazolo[1,5-a]pyrimidine-3-carboxylate (4a) with primary alkylamines 6a-h. Likewise, due to the moderate use of chalcones in PPs syntheses and our interest in accessing this type of heterocycle by eco-compatible methods from easily accessible reagents, 7-10,17 we also proposed obtaining the 5,7diarylsubstituted esters 4a-h by the reaction of 1 with chalcones 2a-h (Scheme 2c). Ultimately, we look for the synthesis of 4a-h and 5a-h through green approaches.
Scheme 2 (a) Synthesis of amides by solid-supported catalysts and using PP esters. (b) Proposal for synthesizing reversan (5h) and its structural analogues.
synthesized from 3-aminocrotononitrile (10) and HM (1.5 equiv.) by a modied method at 120°C for 30 min under microwave; amine 1 ′ was puried by ash chromatography (Scheme 4a). Subsequently, we carried out the syntheses of 1, 2a, and 1 ′ on a scale of about 6, 4, and 3 g, respectively, as they are strategic substrates in our laboratory (Schemes 3 and 4a, data in rectangles). Notably, precursors 1 and 2a were crucial for this work and obtained without chromatographic purication.
In addition, increasing the scale also resulted in a slight increase in yields of 1 (from 85 to 88%), 2a (from 90 to 92%), and 1 ′ (from 79 to 84%). With the required precursors in hand, we envisaged that the reaction of 1 and 2a-h could give 3-carboethoxypyrazolo[1,5-a] pyrimidines 4a-h by the standard route, related to previous works (see Schemes 1 and 2b). In this way, we reproduce the synthesis of 4a ′ by Kaswan et al., 16 where they obtained the product in 82% yield from the amine 1 ′ , chalcone (2a) and KOH as a catalyst in DMF; however, we obtained moderate yield despite the experimental variants used (i.e., time and MW heating). Then, we obtained two other PPs (4b ′ -c ′ ), but the results did not improve; thus, although products can be obtained as reported, this protocol must be revised (Scheme 4b). These results made us question the reactivity of chalcones toward aminoester 1, which is even less nucleophilic than amine 1 ′ .
In general, obtaining the PP esters 4a-h using chalcones 2ag and aminoester 1 is a great challenge due to the reactivity of substrates, the easy access to 2a-g from cheap reagents, and even more due to the absence of a standard method for this synthesis. Thus, we started the study by exploring the reaction of 1 with an equimolar amount of 2c (Ar = 4-ClPhl) to optimize this reaction. We selected 2c due to its high electrophilic character, and the chlorophenyl group generally allows us simple purication processes and follow-up by 1 H NMR. By thin-layer chromatography (TLC), we noted that reactions did not proceed or occur with poor conversion under similar conditions to those used in our laboratory for similar reactions, i.e., without or with polar solvent under microwave, allowing us to carry out several tests quickly. 40 Similar results were evidenced under heating to reux and using non-nucleophilic bases. Decomposition products were obtained by heating the reaction above 180°C (Table 1, entries 1 to 4).
Due to the initial adverse results, we used heterogeneous catalysts such as silica gel, alumina, or NaF/alumina as in the work of Saleh et al. 14 where potassium persulfate (K 2 S 2 O 8 ) was added to favor the oxidation step (Scheme 1a above). The catalytic effect of these solids is based on their diverse acidity (silica < Al 2 O 3 < NaF-Al 2 O 3 ) 41,42 or maybe the NaF-Al 2 O 3 basicity to NH-azoles for the uoride anion. 43 Ester 4c was obtained in poor yields using these solid catalysts (Table 1, entries 5 to 7), but with NaF-Al 2 O 3 , the yields can be increased using a higher F − concentration in the solid or varying the mixture amount; 44 however, the reaction time is crucial for the process (Table 1, entries 7 to 11). These results established that a yield greater than 60% was achieved with catalytic amounts of NaF-Al 2 O 3 ( Table 1, entry 11). The best yield was found when the reaction was heated in fusion using a sand bath at 180°C for 10 min. In the absence of K 2 S 2 O 8 under the optimized conditions, the yield was reduced to 43%, conrming the importance of this oxidizing agent (Table 1, entry 12 vs. 13).
Next, the reaction scope using various chalcones and the optimized conditions was examined. The reaction of an equimolar mixture of 1 with 2a-h (0.5 mmol) in the presence of NaF-Al 2 O 3 (3 : 5 w/w, 25 mg) under heating at 180°C for 10 min and then adding K 2 S 2 O 8 (1 equiv.) to heat for another 5 min at 100°C afforded the novel family of ethyl-5,7-diarylpyrazolo[1,5-a] pyrimidine-3-carboxylates 4a-h in high yields. Notably, substrates 2a-f have the same aryl group, which differs in 2g-h; this feature allows us to better establish the reaction regioselectivity in the initial step to form PPs 4a-h (Scheme 5).
Almost no loss of efficiency was observed in the synthesis of 4a-h with the chalcones tested, evidencing that the electronic demands of the substituents had little inuence on the reactivity beyond the possible decomposition or evaporation of reagents under the established reaction conditions. However, the lowest yields were obtained using chalcones 2g-h, possibly due to the formation of 4h (using 2g) and 4g (using 2h), which are regioisomers of esters 4g and 4h, respectively (Scheme 5, blue rectangle); in any case, the high regioselectivity of reactions was demonstrated. Due to ester 4a being the key precursor for synthesizing the nal products in this work (i.e., Reversan and analogue amides), we carried out its synthesis on a scale of 2 g (Schemes 5, green rectangle). In this case, increasing the scale also resulted in a slight increase in the yields of 4a (from 81 to 84%).
Although there are reports for preparing PPs via the reaction of NH-5-aminopyrazoles with chalcones, 14-17 a reasonable reaction route to yield products 4a-h under the optimized conditions in this work was established (Scheme 5 at the top). The reaction begins with an aza-Michael addition of the pyrrolic-like nitrogen atom in 1 (N1) to the Cb of 2a-f, leading to intermediate I through a typical and dominant so-so interaction (Fig. 2). 11,45,46 This attack is probably favored by uoride ions (F − ) in the catalyst, having a veried ability to remove the hydrogen atom from NH-azoles; 43 likewise, the enone increases its electrophilicity when the carbonyl group interacts with alumina. Then, the cyclcondensation of I with the loss of a water molecule occurs to afford the dihydro derivative II (NH 2 /hard / C]O/hard), which is oxidized to the product 4a-h with K 2 S 2 O 8 (1 equiv.) that favors the removal of hydrogen by converting to potassium bisulfate (KHSO 4 ). 14 Gratifyingly, structures of compounds 4a and 4g were solved by single-crystal X-ray diffraction analysis (see ESI † † for details). These results allowed us to verify the reaction course since substrate 2g, which leads to 4g, has two different aryl groups. It was impossible to establish the regioselectivity of the cyclocondensation reaction only using NMR analysis such as NOESY or HMBC experiments (Fig. 2). It is important to mention what was cited in the introduction section: only four articles on obtaining 5,7-diarylpyrazolo[1,5-a] pyrimidines 4 starting from aminoester 1 have been published. [20][21][22][23] In fact, only one of these works is comparable with synthesizing the eight PPs 4a-h because chalcones were used as substrates, 22 and only compounds 4a and 4f have been reported in the literature. However, the method developed in this research presented better results than the cited works regarding process efficiency, synthetic versatility, and substrate scope (e.g., chalcones or enones are easily accessible both synthetically and cheaply) and/or yields (see Scheme 1 above). [20][21][22][23] Synthesis of Reversan and analogues 5a-h Once PPs 4a-h were obtained, we selected compound 4a to develop a simple and green method to generate Reversan and its structural analogues 5a-h by the direct amidation reaction of this ester with primary alkylamines 6a-h. In the amides synthesis on PPs, the respective carboxylic acid is used as a precursor; however, due to the possibility of the direct amidation of ester 4a, we tried to synthesize the N-butycarboxamide 5b with n-butylamine (2b) as a model reagent that allows us to optimize the conditions for this transformation, saving the hydrolysis step, although this reaction type is minorly used. Thus, considering our interest in MW-mediated reactions 40 and the results of the NaF/alumina-catalyzed synthesis of 4a-g, we decided to start the study using similar conditions to those described by Ojeda-Porras et al.; 33 they achieved the amidation of carboxylic acids with amines by irradiating an equimolar mixture (1.5 mmol) of reagents supported on silica gel (1.0 g) with MW for 4 cycles of 20 min at 130°C (see Scheme 2a above). However, our results were not encouraging even aer increasing the time and temperature ( Table 2, entries 1 to 3).
Because 6b is a volatile amine, we decided to double its proportion (from 0.15 to 0.30 mmol), wanting this would favor the reaction; however, it was only possible to observe the forming 5b in moderate yield aer 1 hour of reaction at 180°C ( Table 2, entries 4 to 7). Next, we tripled the proportion of 6b (0.45 mmol), allowing us to form 5b in good yields, but the reaction is not better at less than 180°C or more than 30 min ( Table 2, entries 8 to 10).
Consequently, the optimal reaction conditions to obtain 5b use ∼3 equivalents of starting amine 6b and 0.1 g silica gel at 180°C for 30 min under microwave irradiation (Table 2, entry 9); we employed these conditions to examine the amidation reaction scope with various commercial primary alkylamines 6a-h. Fortunately, the MRP1 inhibitor, Reversan (5h), was obtained by the reaction of 4h with 3-morpholinopropanamine (6h); likewise, using alkylamines 6a-g, the Reversan structural analogues 5a-g were obtained, and all the synthesized amides were obtained in high yields. Moreover, crystals of suitable size and quality for single-crystal X-ray diffraction analysis of products 5a (N-iPr), 5d (N-Bn), and 5h (Reversan) were obtained by their recrystallization from methanol/ethyl acetate (1 : 3) using the slow evaporation method (Scheme 6, see ESI † for details).
Remarkable, silica gel is a heterogeneous catalyst serving challenging reactions that an acid medium could favor, such as the amidation reaction of esters studied in this work (see Scheme 6, at the top). In addition, due to the biological relevance and the high price of Reversan (5h), we obtained it on a one gram scale in high yield (80%) using 3.0 mmol ester 4a and 9 mmol 6h (Scheme 6, data in rectangle). Thus, this synthesis is a valuable input to the scientists investigating drug discovery with biological, pharmacological, or medicinal applications because we used cheap substances (i.e., 3-morpholinopropanamine (6h), ethyl acetoacetate (7), acetophenone (8a), benzaldehyde (9a), DMF-DMA, acetic acid, hydrazine, KOH, NaF, K 2 S 2 O 8 , alumina, and silica gel) through simple, highly efficient, and even scalable synthetic methodologies (Scheme 7). Finally, pyrazolo[1,5-a]pyrimidine ester 4a was also obtained by the reaction of dibenzoylmethane (3) 23 with pyrazole aminoester 1 in microwaves, allowing us to develop a second synthesis of Reversan (route B) to compare with the rst synthesis through enone 2a (route A). In route A, an overall yield of 54.4% was obtained in four reaction steps, while route B proceeded in three reaction steps with an overall yield of 38.7% using substrate 3, which is relatively expensive and more difficult to prepare than 2a (Scheme 7). 23 Consequently, route A is better than route B because the best global yield is obtained, and with compounds 4b-h, greater structural diversity can be obtained for further studies related to Reversan and its structural analogues.
As a nal comment regarding the results of this investigation, it should be noted that only one (Reversan 5h) of the eight amides synthesized 4a-h has been reported in the literature, and as cited in the introduction, a synthetic route for Reversan, which documents all the syntactic and characterization details, is yet to be reported.

Conclusion
In summary, Reversan (5h) and its structural analogues (amides 5a-g) were synthesized by the silica-mediated direct amidation reaction between ethyl-5,7-diphenylpyrazolo[1,5-a]pyrimidine-3-carboxylate (4a) and primary amines 6a-h. In addition, a family of ethyl-5,7-diarylpyrazolo[1,5-a]pyrimidine-3carboxylates 4a-h was obtained when aminoester 1 was cyclocondensed with chalcones 2a-h using the NaF-alumina catalyst and as the nal step, a Na 2 S 2 O 8 -mediated oxidation reaction. All products were obtained in high yields via simple, efficient, and scalable methodologies using cheap reagents, heterogeneous catalysts such as silica gel or NaF-alumina, and solvent-free reactions in fusion or microwaves. Remarkably, the two relevant reaction types for this work (cyclocondensations of chalcones with 5-aminopyrazoles bearing an EWG and esters amidation) are protocols rarely used. In addition, the obtained compounds were characterized by spectroscopic analysis, and the structures of some intermediates and products (4a, 4g, 5a, 5d, and 5h) were conrmed by single-crystal X-ray diffraction analysis. Therefore, we developed synthetic methods that address some of the key points associated with green chemistry principles employing easily accessible substances.

Reagents and materials
The reagents and substances used in this investigation were purchased from commercial sources and used without further purication; these were weighed and handled in the air at room temperature. The reaction was monitored by thin-layer chromatography (TLC), visualized by a UV lamp (254 or 365 nm), and ash chromatography was performed on silica gel (230-400 mesh). Reactions under microwave irradiation were carried out in a sealed reaction vessel (10.0 mL, max pressure = 300 psi) containing a Teon-coated stir bar (obtained from CEM) and were performed in a CEM Discover SP-focused MW (n = 2.45 GHz) reactor equipped with a built-in pressure measurement sensor and a vertically-focused IR temperature sensor. Controlled temperature, power, and time settings were used. Substrates based on NH-5-aminopyrazoles 1 and 1 ′ and chalcone derivatives 2a-h were prepared by known procedures and developed by us (see ESI † for details of the synthesis of these substrates).
The NMR spectra for this work were recorded at 400 MHz ( 1 H) and 101 MHz ( 13 C) at 298 K, and the data were recorded in CDCl 3 (7.26/77.05 ppm) or DMSO (2.50/39.5 ppm) using the residual nondeuterated signal for 1 H and the deuterated solvent signal for 13 C NMR as internal standards. Chemical shis (d) are given in parts per million (and coupling constants (J) in Hertz (Hz). The multiplicity abbreviations involve s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet (see copies of NMR spectra in Fig. S3-S31 of ESI †). Melting points were determined using a capillary melting point apparatus, and the data were uncorrected. High-resolution mass spectra (HRMS) were recorded using a Q-TOF spectrometer by electrospray ionization (ESI) (see the HRMS analysis in Fig. S32-S47 of ESI †). The X-ray intensity data were measured at 25(2)°C using CuKa radiation (l = 1.54184 Å), by u scans in an Agilent SuperNova, Dual, Cu at Zero, Atlas four-circle diffractometer equipped with a CCD plate detector (see ESI † for more crystallographic details).

Synthesis and characterization
Synthesis of 5,7-diarylpyrazolo[1,5-a]pyrimidines 4a ′ -c ′ . A mixture of 3-methyl-1H-pyrazol-5-amine (1 ′ , 0.6 mmol, 58 mg), the respective chalcone derivative 2 (0.5 mmol), KOH (0.05 mmol 3 mg), and DMF (2.5 mL) was heated at 110°C for 20 min under constant stirring. The mixture was then allowed to cool to room temperature, water (10 mL) was added, and the aqueous mixture was extracted with ethyl acetate (3 × 10 mL). The organic extract was dried over anhydrous Na 2 SO 4 , and the solvent was removed under reduced pressure. The residue was puried by ash chromatography on silica gel (eluent: npentane/AcOEt 4 : 1 v/v) to afford products 4a ′ -d ′ (ref. 16 13 (C) ppm. These NMR data matched previously reported data. 16 Synthesis of 3-carboethoxypyrazolo[1,5-a]pyrimidines 4a-h. Equimolar amounts of ethyl-5-amino-1H-pyrazole-4-carboxylate (1, 0.5 mmol, 78 mg) and the respective enone 2a-h were thoroughly mixed at room temperature, together with 25 mg of NaF-Al 2 O 3 (3 : 5, w/w), into a 10 mL sealable (Teon screw cap) tubular reaction vessel. The mixture was heated in fusion over the solid support and catalyst at 180°C for 10 min using a sand bath. Next, 1 equiv. K 2 S 2 O 8 (135 mg) was added, and the mixture continued heating for another 5 min at 100°C. The resulting reaction mixture was then cooled to room temperature and extracted with ethyl acetate (3 × 10 mL). The extract was dried over anhydrous Na 2 SO 4 , the solvent was removed, and the crude product was puried by ash chromatography on silica gel (eluent: CH 2 Cl 2 ) to afford the desired products 4a-h in good yields. The recrystallization of 4a and 4g from methanol/AcOEt (1 : 3) afforded crystalline colorless prisms suitable for X-ray diffraction analysis.